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Neglected tropical diseases are those caused by infectious agents or parasites and are considered endemic in low-income populations. These diseases also have unacceptable indicators and low investment in research, drug production, and control. Tropical diseases such as leishmaniasis are some of the main causes of morbidity and mortality around the globe. Electrochemical immunosensors are promising tools for diagnostics against these diseases. One such benefit is the possibility of assisting diagnosis in isolated regions, where laboratory infrastructure is lacking. In this work, different peptides were investigated to detect antibodies against Leishmania in human and canine serum samples. The peptides evaluated (395-KKG and 395-G) have the same recognition site but differ on their solid-binding domains, which ensure affinity to spontaneously bind to either graphene oxide (GO) or graphene quantum dots (GQD). Cyclic voltammetry and differential pulse voltammetry were employed to investigate the electrochemical behavior of each assembly step and the role of each solid-binding domain coupled to its anchoring material. The graphene affinity peptide (395-G) showed better reproducibility and selectivity when coupled to GQD. Under the optimized set of experimental conditions, negative and positive human serum samples responses were distinguished based on a cut-off value of 82.5% at a 95% confidence level. The immunosensor showed selective behavior to antibodies against Mycobacterium leprae and Mycobacterium tuberculosis, which are similar antibodies and potentially sources of false positive tests. Therefore, the use of the graphene affinity peptide as a recognition site achieved outstanding performance for the detection of Leishmania antibodies.

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The objective of these studies was to evaluate the inclusion of a microbial muramidase (MUR) in the diets of broiler chickens on the growth performance, intestinal permeability (IP), total blood carotenoid content, apparent ileal digestibility (AID), and foot pad dermatitis (FPD). In Experiment 1, a total of 1,000 one-day-old chicks were placed in floor-pens with reused litter, and randomly distributed into 4 treatments with 10 replicates each. Treatments were a basal diet (control), or basal diet supplemented with 15,000; 25,000 or 35,000 LSU (F)/kg of MUR. Feed intake (FI), body weight gain (BWG), and feed conversion ratio (FCR) were evaluated at d 21 and 43. Intestinal permeability was evaluated on d 35 by FITC-d, and FPD and AID on d 43. In Experiment 2, a total of 800 one-day-old chicks were placed in floor-pens with fresh litter, and randomly distributed into 4 treatments with 8 replicates each. Treatments were a basal diet (control), or basal diet supplemented with 25,000 or 35,000 LSU (F)/kg of MUR, and a fourth group where the basal diet was supplemented with enramycin. The birds were induced to a mild intestinal challenge. Feed intake, BWG, and FCR were evaluated on d 21 and d 42, and total blood concentration of carotenoids was evaluated on d 28. In experiment 1, 35,000 LSU (F)/kg of MUR promoted the best FCR (P < 0.05). Muramidase supplementation linearly increased the AID of dry matter, ash, and fat (P < 0.01), and regardless of the dose, MUR decreased the IP (P < 0.05). In Experiment 2, the supplementation of 35,000 LSU (F)/kg of MUR improved BWG and FCR in the entire cycle (1-42 d) and increased the concentration of carotenoids in the blood on d 28 compared to the control group (P < 0.05). These studies show that MUR improves growth performance of broilers by improving intestinal permeability, digestibility of dry matter, ash and fat, absorption of carotenoids, and reducing FPD.

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The development of immunosensors to detect antibodies or antigens has stood out in the face of traditional methods for diagnosing emerging diseases such as the one caused by the SARS-CoV-2 virus. The present study reports the construction of a simplified electrochemical immunosensor using a graphene-binding peptide applied as a recognition site to detect SARS-CoV-2 antibodies. A screen-printed electrode was used for sensor preparation by adding a solution of peptide and reduced graphene oxide (rGO). The peptide-rGO suspension was characterized by scanning electron microscopy (SEM), Raman spectroscopy, and Fourier transform infrared spectroscopy (FT-IR). The electrochemical characterization (electrochemical impedance spectroscopy-EIS, cyclic voltammetry-CV and differential pulse voltammetry-DPV) was performed on the modified electrode. The immunosensor response is based on the decrease in the faradaic signal of an electrochemical probe resulting from immunocomplex formation. Using the best set of experimental conditions, the analytic curve obtained showed a good linear regression (r2 = 0.913) and a limit of detection (LOD) of 0.77 µg mL-1 for antibody detection. The CV and EIS results proved the efficiency of device assembly. The high selectivity of the platform, which can be attributed to the peptide, was demonstrated by the decrease in the current percentage for samples with antibody against the SARS-CoV-2 S protein and the increase in the other antibodies tested. Additionally, the DPV measurements showed a clearly distinguishable response in assays against human serum samples, with sera with a response above 95% being considered negative, whereas responses below this value were considered positive. The diagnostic platform developed with specific peptides is promising and has the potential for application in the diagnosis of other infections that lead to high antibody titers.

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In the present work, we report an innovative approach for immunosensors construction. The experimental strategy is based on the anchoring of biological material at screen-printed carbon electrode (SPE) modified with electrodeposited Graphene Quantum Dots (GQD) and polyhydroxybutyric acid (PHB). It was used as functional substract basis for the recognition site receptor-binding domain (RBD) from coronavirus spike protein (SARS-CoV-2), for the detection of Anti-S antibodies (AbS). SEM images and EDS spectra suggest an interaction of the protein with GQD-PHB sites at the electrode surface. Differential pulse voltametric (DPV) measurements were performed before and after incubation, in presence of the target, shown a decrease in voltametric signal of an electrochemical probe ([Fe(CN)6]3/4-). Using the optimal experimental conditions, analytical curves were performed in PBS and human serum spiked with AbS showing a slight matrix effect and a relationship between voltametric signal and AbS concentration in the range of 100 ng mL-1 and 10 µg mL-1. The selectivity of the proposed sensor was tested against yellow fever antibodies (YF) and the selective layer on the electrode surface did not interact with these unspecific antibodies. Eight samples of blood serum were analyzed and 87.5% of these total investigated provided adequate results. In addition, the present approach showed better results against traditional EDC/NHS reaction with enhancements in time and the possibility to develop an immunosensor in a single drop, since the proteins can be anchored prior to the electrode modification step.

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Erysipelothrix sp. isolates obtained from a deadly outbreak in farmed turkeys were sequenced and compared to representatives of the genus. Phylogenetic trees-supported by digital DNA:DNA hybridization and Average Nucleotide Identity-revealed a novel monophyletic clade comprising isolates from pigs, turkeys, and fish, including isolates previously described as E. sp. Strain 2. Genes coding for the SpaC protein, typically found in E. sp. Strain 2, were detected in all isolates of the clade. Therefore, we confirm E. sp. Strain 2 represents a unique species that may be isolated from a broad host range, and the name "Erysipelothrix takahashiae" is suggested. Core genome analysis showed that the pathogenic species of this genus, E. rhusiopathiae and the clade E. sp. Strain 2, are enriched in core functionalities related to nutrient uptake and transport, but not necessarily homologous pathways. For instance, whereas the aerobic DctA transporter may uptake C4-dicarboxylates in both species, the anaerobic DcuC transporter is exclusive of the E. sp. Strain 2. Remarkably, the pan-genome analysis uncovered that genes related to transport and metabolism, recombination and repair, translation and transcription in the fish isolate, within the novel clade, have undergone a genomic reduction through pseudogenization. This reflects distinct selective pressures shaping the genome of species and strains within the genus Erysipelothrix while adapting to their respective niches.

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Probiotics and immunization are being widely adopted by the poultry industry with the goal of controlling Salmonella enterica. However, the interaction between these two management protocols has been sparsely studied. The present study aimed to understand the role of an Enterococcus faecium probiotic in the production of salmonella-specific IgA in layers immunized with a live vaccine. Four groups were used: "Control" (no vaccine or probiotic); "Probiotic" (which received an E. faecium product); "Vaccine" (immunized with two doses of a live attenuated S. Enteritidis vaccine); and "Vaccine + probiotic". Faecal salmonella-specific IgA was analysed 7 and 20 days post-vaccination (dpv) boost. At 7 dpv, the "Vaccine" and "Vaccine + probiotic" groups had similar IgA levels. However, at 20 dpv, IgA levels were two times higher in the "Vaccine + probiotic" group compared to the "Vaccine" group. To understand the role of the intestinal microbiota in this finding, bacterial diversity in faeces was analysed by 16S rRNA gene sequencing. The improvement in IgA production in probiotic-treated birds was accompanied by marked changes in the faecal microbiome. Some of the main differences between the "Vaccine" and "Vaccine + probiotic" groups included reduction of Escherichia-Shigella and increases in Blautia, Anaerotruncus and Lactobacillus in the latter group. Although no direct causal link can be established from this study design, it is possible that the E. faecium probiotic induces improved antibody production following vaccination via modulation of the intestinal microbiota.

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A Salmonelose é uma importante zoonose, considerada a principal causa de infecções bacterianas, sendo associada ao consumo de produtos avícolas. Como alternativa de controle, ácidos orgânicos têm sido amplamente usados. No entanto, pouco se conhece sobre o estado imunológico de aves de produção, e uma avaliação deste status é necessária para proteger frente a enfermidades e para garantir à aplicação segura de agentes terapêuticos ou imunização profilática. Este trabalho teve como objetivo verificar o comportamento do sistema imunológico das aves previamente infectadas com Salmonella Enteritidis (SE) tratadas com um composto de ácidos orgânicos em diferentes concentrações administrado via água e ração comparando com as aves infectadas e não tratadas. Foram inoculados 120 frangos de corte com 1mL de SE, via oral, na concentração de 1,0 x 108 UFC/mL, no 1º e 2º dia de idade, divididos em seis tratamentos com duas repetições, utilizando 200, 400, 500 e 1000ppm do ácido orgânico. Aos 35 dias de vida das aves, foram coletados, de todos os grupos, alíquotas de sangue de 3mL em tubo contendo EDTA para a avaliação das células imunes através de citometria de fluxo. Foram analisadas as porcentagens circulantes de células CD4+, CD8β+, MHC I+, MHC II+, TCRVβ1+, TCRVβ2+ e CD28+. Para análise microbiológica foram coletadas tonsilas cecais destas aves. Observou-se com esse estudo que os ácidos orgânicos nas dosagens 1000ppm na água e 500ppm na ração durante, dois e sete dias respectivamente antes do abate, foram eficazes na redução da infecção por SE em frangos de corte, comprovadas pelo método microbiológico e demonstradas através do comportamento das células do sistema imune. No presente estudo as aves infectadas apresentaram uma proporção menor de células T auxiliares circulantes quando comparadas às aves infectadas, mas tratadas com o AO ou com o grupo não infectado. A mesma tendência pode ser observada para as células CD28+, TCRVβ1+ e MHC IIbright+, e, com menor resolução, para CD8β+.(AU)

Salmonellosis is an important zoonosis, considered the leading cause of bacterial infections, and is associated with the consumption of poultry products. As alternative control, organic acids have been widely used. However, little is known about the immune status of poultry production, and an evaluation of this status is necessary to protect against disease and to ensure the safe application of therapeutic agents or prophylactic vaccination. This study aimed to verify the behavior of the immune system of birds previously infected with Salmonella Enteritidis (SE) treated with a compound of organic acids in different concentrations administered via water and food, compared with the infected birds and untreated. One hundred and twenty broilers were orally inoculated with 1ml of SE at a concentration of 1.0x108 CFU/mL, at 1 and 2-days-old and divided into six treatments with two repetitions of 200, 400, 500 and 1000ppm organic acid. From 35-days-old birds of all groups were collected aliquots of 3mL of blood into a tube containing EDTA for the evaluation of immune cells by flow cytometry. We then analyzed the percentages of circulating CD4+, CD8β+, MHC I+ MHC II+, TCRVβ1+, CD28+ + and TCRVβ2. For microbiological analysis were collected caecal tonsils of these birds. We found that organic acids in dosages 1000ppm 500ppm in water and in feed for 2 to 7 days before slaughter, respectively, were effective in reducing SE infection in broilers, proven by microbiological method and demonstrated through the behavior of immune cells. The infected birds showed a lower proportion of circulating T helper cells compared with infected poultry, but treated with AO or with the uninfected group. The same trend can be observed for CD28+ cells, and MHC IIbright+ TCRVβ 1+, and with lower resolution, for CD8β+.(AU)

Assuntos

RESUMO

A Salmonelose é uma importante zoonose, considerada a principal causa de infecções bacterianas, sendo associada ao consumo de produtos avícolas. Como alternativa de controle, ácidos orgânicos têm sido amplamente usados. No entanto, pouco se conhece sobre o estado imunológico de aves de produção, e uma avaliação deste status é necessária para proteger frente a enfermidades e para garantir à aplicação segura de agentes terapêuticos ou imunização profilática. Este trabalho teve como objetivo verificar o comportamento do sistema imunológico das aves previamente infectadas com Salmonella Enteritidis (SE) tratadas com um composto de ácidos orgânicos em diferentes concentrações administrado via água e ração comparando com as aves infectadas e não tratadas. Foram inoculados 120 frangos de corte com 1mL de SE, via oral, na concentração de 1,0 x 108 UFC/mL, no 1º e 2º dia de idade, divididos em seis tratamentos com duas repetições, utilizando 200, 400, 500 e 1000ppm do ácido orgânico. Aos 35 dias de vida das aves, foram coletados, de todos os grupos, alíquotas de sangue de 3mL em tubo contendo EDTA para a avaliação das células imunes através de citometria de fluxo. Foram analisadas as porcentagens circulantes de células CD4+, CD8β+, MHC I+, MHC II+, TCRVβ1+, TCRVβ2+ e CD28+. Para análise microbiológica foram coletadas tonsilas cecais destas aves. Observou-se com esse estudo que os ácidos orgânicos nas dosagens 1000ppm na água e 500ppm na ração durante, dois e sete dias respectivamente antes do abate, foram eficazes na redução da infecção por SE em frangos de corte, comprovadas pelo método microbiológico e demonstradas através do comportamento das células do sistema imune. No presente estudo as aves infectadas apresentaram uma proporção menor de células T auxiliares circulantes quando comparadas às aves infectadas, mas tratadas com o AO ou com o grupo não infectado. A mesma tendência pode ser observada para as células CD28+, TCRVβ1+ e MHC IIbright+, e, com menor resolução, para CD8β+.

Salmonellosis is an important zoonosis, considered the leading cause of bacterial infections, and is associated with the consumption of poultry products. As alternative control, organic acids have been widely used. However, little is known about the immune status of poultry production, and an evaluation of this status is necessary to protect against disease and to ensure the safe application of therapeutic agents or prophylactic vaccination. This study aimed to verify the behavior of the immune system of birds previously infected with Salmonella Enteritidis (SE) treated with a compound of organic acids in different concentrations administered via water and food, compared with the infected birds and untreated. One hundred and twenty broilers were orally inoculated with 1ml of SE at a concentration of 1.0x108 CFU/mL, at 1 and 2-days-old and divided into six treatments with two repetitions of 200, 400, 500 and 1000ppm organic acid. From 35-days-old birds of all groups were collected aliquots of 3mL of blood into a tube containing EDTA for the evaluation of immune cells by flow cytometry. We then analyzed the percentages of circulating CD4+, CD8β+, MHC I+ MHC II+, TCRVβ1+, CD28+ + and TCRVβ2. For microbiological analysis were collected caecal tonsils of these birds. We found that organic acids in dosages 1000ppm 500ppm in water and in feed for 2 to 7 days before slaughter, respectively, were effective in reducing SE infection in broilers, proven by microbiological method and demonstrated through the behavior of immune cells. The infected birds showed a lower proportion of circulating T helper cells compared with infected poultry, but treated with AO or with the uninfected group. The same trend can be observed for CD28+ cells, and MHC IIbright+ TCRVβ 1+, and with lower resolution, for CD8β+.

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RESUMO

Dois experimentos foram desenvolvidos para avaliar a eficiência de ácidos orgânicos frente a Salmonella enterica enterica sorovar Enteritidis (SE) e Minnesota (SM) em frangos. No primeiro experimento foram avaliados 3 tratamentos: T1 - ração adicionada de ácido orgânico, T2 - ração adicionada de ácido orgânico e ácido orgânico na água de bebida, T3 - grupo controle. Todos os animais foram inoculados com SE, via oral. A utilização de ácidos orgânicos na ração (T1) e na ração e na água (T2) diminuíram a excreção de Salmonella no papo e no ceco 7 dias pós inoculação com SE e houve redução de células CD3+ no jejuno dos frangos. No segundo experimento foram avaliados 4 tratamentos sendo T1 - controle, T2 - controle inoculado via oral com Salmonella Minnesota (SM), T3 - animais inoculados via oral com SM e ácidos orgânicos na ração e T4 - animais inoculados via oral com SM e ácidos orgânicos na ração e na água de bebida. Ácidos orgânicos a ração (T3) e na ração e na água (T4) reduziram a excreção de SM em papo de frangos de corte desafiados, 7 dias após inoculação. O uso de ácidos orgânicos na ração e na ração e na água foram mais eficientes em reduzir SE do que SM.(AU)

Two experiments were carried out to evaluate effectiveness of organic acids against Salmonella enterica enterica serovars Enteritidis (SE) and Minnesota (SM) in broilers. In the first experiment three treatments were evaluated: T1 - feeding with organic acids, T2 - feeding with organic acids and organic acids in drink water, and T3 - control group. All animals were oral challenged with SE. Organic acids in diet (T1) and organic acids in diet and drink water (T2) reduced the shadding of Salmonella in crop and cecum 7 days post challenged with SE and reduced the CD3+ cells in jejunal mucosa of broilers. In the second experiment four treatments were evaluated, T1 - control group, T2 - control group oral challenged with Salmonella Minnesota (SM); T3 - oral challenged animals with SM and treated with organic acids in diet; T4 - oral challenged animals with SM and treated with organic acids in diet and in drink water. Organic acids in diet (T3) and organic acids in diet and in drink water (T4) reduced the shadding of SM in crop of challenged broilers, 7 days post inoculation The use of organic acids in diet and in water was more effective to control SE than SM.(AU)

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RESUMO

Dois experimentos foram desenvolvidos para avaliar a eficiência de ácidos orgânicos frente a Salmonella enterica enterica sorovar Enteritidis (SE) e Minnesota (SM) em frangos. No primeiro experimento foram avaliados 3 tratamentos: T1 - ração adicionada de ácido orgânico, T2 - ração adicionada de ácido orgânico e ácido orgânico na água de bebida, T3 - grupo controle. Todos os animais foram inoculados com SE, via oral. A utilização de ácidos orgânicos na ração (T1) e na ração e na água (T2) diminuíram a excreção de Salmonella no papo e no ceco 7 dias pós inoculação com SE e houve redução de células CD3+ no jejuno dos frangos. No segundo experimento foram avaliados 4 tratamentos sendo T1 - controle, T2 - controle inoculado via oral com Salmonella Minnesota (SM), T3 - animais inoculados via oral com SM e ácidos orgânicos na ração e T4 - animais inoculados via oral com SM e ácidos orgânicos na ração e na água de bebida. Ácidos orgânicos a ração (T3) e na ração e na água (T4) reduziram a excreção de SM em papo de frangos de corte desafiados, 7 dias após inoculação. O uso de ácidos orgânicos na ração e na ração e na água foram mais eficientes em reduzir SE do que SM.

Two experiments were carried out to evaluate effectiveness of organic acids against Salmonella enterica enterica serovars Enteritidis (SE) and Minnesota (SM) in broilers. In the first experiment three treatments were evaluated: T1 - feeding with organic acids, T2 - feeding with organic acids and organic acids in drink water, and T3 - control group. All animals were oral challenged with SE. Organic acids in diet (T1) and organic acids in diet and drink water (T2) reduced the shadding of Salmonella in crop and cecum 7 days post challenged with SE and reduced the CD3+ cells in jejunal mucosa of broilers. In the second experiment four treatments were evaluated, T1 - control group, T2 - control group oral challenged with Salmonella Minnesota (SM); T3 - oral challenged animals with SM and treated with organic acids in diet; T4 - oral challenged animals with SM and treated with organic acids in diet and in drink water. Organic acids in diet (T3) and organic acids in diet and in drink water (T4) reduced the shadding of SM in crop of challenged broilers, 7 days post inoculation The use of organic acids in diet and in water was more effective to control SE than SM.

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